Structure and function of the murine muscle Уtendon junction.

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THE ANATOMICAL RECORD 201293-302 (1981)
Structure and Function of the Murine Muscle-Tendon Junction
JOHN A. TROTTER, KAREN CORBETT, AND BARRY P. AVNER
Department of Anatomy (JA.T.,K.C.) and Department of Pharmacology ( B P A J ,
University of New Mexico School of Medicine, Albuquerque, NM 87131
ABSTRACT
The muscle-tendon junctions of the extensor carpi radialis long u s and brevis muscles from adult Balb C BaileyN mice have been examined
tensiometrically and ultrastructurally following removal of cellular membrane
and soluble cytoplasm by exposure to nonionic detergent. As judged by the ability
of the extracted muscle to generate tension upon exposure to ATP and to transmit
the generated tension to the tendon, detergent extraction leaves the
muscle-tendon junction functionally intact. Electron microscopic analysis of the
extracted muscle-tendon junctions reveals that the relationship between the terminal myofilaments and the lamina densa of the basal lamina is retained, despite
the extensive extraction of the plasma membrane. Fine filaments (2-7 nm) are
seen to connect the lamina densa with an electron-dense intracellular layer into
which terminal actin filaments appear to insert. These fine filaments are considered to represent an important component of the structural linkage between
myofilaments and connective tissue and hence to be a significant component of
the tension transmitting mechanism. Their precise nature is not known, but some
part of the filaments must pass through the hydrophobic compartment of the
plasma membrane and thus must be a transmembrane component of considerable
tensile strength. These studies suggest that detergent-extractable membrane lipids play no significant role in the transmission of tension at the muscle-tendon
junction, and that fine filaments, probably protein, are responsible for transmitting tension from myofilaments, through the plasma membrane, to the lamina
densa of the basal lamina.
Early research concerning the mechanism
by which force generated within a skeletal
muscle fiber is transmitted to the tendon concentrated on two questions: (a) does force
transmission occur only at the ends of the muscle fiber, or does mechanical coupling occur all
along the fiber; and (b) does the plasma membrane form a continuous boundary between
sarcoplasm and tendon, or are myofilaments
continuous with tendon filaments at the myotendinous junction. Modern work supports
the first alternative in each case (Bennett,
1955; Gelber et al., 1960). In particular, the
electron microscope has revealed that a continuous bilayer membrane forms the external
boundary of the cytoplasm of skeletal muscle
cells, as it does of all other cells. The presence
of a morphologically recognizable membrane
implies that a continuous double layer of lipid
(including phospholipid, glycolipids, and cholesterol) separates myofilaments from connective tissue components.
Lipids, however, are characterized by low
shear and tensile strength. Since skeletal muscle fibers can generate forces of approximately
lo6 dyn/cm2, and the tensile strength of collagen is greater than lo8 dyn/cm2(Alexander,
1968), it is likely that some nonlipid component of the membrane must transmit force
across the lipid domain. The only known nonlipid macromolecular species which spans the
thickness of natural membranes is protein.
These considerations strongly suggest the hypothesis that transmembrane proteins are essential links between myofilaments and connective tissue filaments.
Received February 2, 1981: accepted May 6, 1981
294
J.A. TROTTER, K. CORBETT, AND B.P. AVNER
Previous work from this laboratory (Trotter
et al., 1978) and others has demonstrated that
nonionic detergents can be effectively employed to dissolve many membrane lipids because of their ability to act as lipid solvents by
disrupting hydrophobic bonds (Helenius and
Simms, 1975). At the same time, nonionic detergents are largely ineffective in disrupting
most protein-protein interactions. In particular, the nonionic detergent Triton X-100 has
been shown to maintain the contractile apparatus of both striated (Solaro et al., 1971) and
smooth muscle (Small, 1977; Gordon, 1978) in
a functionally intact state. Similarly, Triton
X-100 has no known effects on the protein or
polysaccharide components of the connective
tissues.
In a n effort to test the hypothesis that transmembrane proteins are essential mechanical
links between myofilaments and connective
tissue filaments, the present studies were undertaken to determine: (a) whether the lipid
portion of the plasma membrane of the myotendinous junction can be extracted by Triton
X-100; (b) what effect extraction of membrane
lipids by Triton X-100 would have on the mechanical properties of the myotendinous junction; and (c) what are the ultrastructural characteristics
of
the
detergent-extracted
myotendinous junction.
MATERIALS AND METHODS
Detergent extraction of muscle-tendon
preparations
Extensor carpi radialis longus and brevis
muscles were dissected from adult Balb C BaileylJ mice under sodium pentothal-induced
anesthesia. The exposed tissues were maintained in a moist condition by irrigation with
physiologic saline (see below). Prior to resection, the muscles were tied distally by their
tendons and proximally by their muscle bellies
to 0.5-mm diameter tungsten wire, using 000
silk sutures. The muscles were then severed
at their origins and insertions. The muscles
tied to tungsten wires were extracted for 48 h r
in ice-cold extraction solution. For the physiological experiments, the extraction solution
contained EGTA. For morphological examination the extraction solution contained
EGTA, CaCI,, or neither. (The exact compositions of the extraction solutions and the composition of abbreviated substances are given
below.) They were then rinsed in a n identical
solution without PMSF or Triton X-100. In
some experiments, both sutures were tied to
muscle (i.e., the distal suture was tied proximal to the myotendinousjunction), or both sutures were tied to the same tendon (i.e., the
proximal suture was tied distal to the myotendinous junction). In this series of experiments
41 muscles were extracted and analyzed as
described below.
Determination of isometric tension generated
by extracted muscles
Extracted and rinsed muscles were suspended by the sutures in a tissue bath containing 10 ml of equilibration solution (see
below) at 30°C, with one suture fixed to the
bottom of the chamber and the other connected
to a Grass model FT.03 force displacement
transducer. The initial tension on each muscle
was adjusted to 0.5g and maintained at that
level during the equilibration period of 20-30
min. Nitrogen gas was bubbled through the
solution throughout the experiment. Contraction was initiated by the addition of 0.1 M ATP
and 1 M CaC1, to the final concentrations indicated in the “contracting solution” below.
Mechanical responses were recorded isometrically on a Grass model 7 polygraph.
Fixation a n d microscopy
Fresh and extracted muscles were fixed by
immersion in 2.4% glutaraldehyde, 0.1 M sodium phosphate buffer, pH 7.0, at room temperature for 18 hr. They were then rinsed in
0.1 M phosphate buffer, and postfixed in icecold 1%Os04, 0.1 M phosphate buffer, pH 6.0,
for 60 min. After rinsing in water for 30 min,
the muscles were immersed in 0.5% uranyl
acetate in water for 60 min, and were then
dehydrated in a series of increasing concentrations of ethanol. The ethanol was replaced
by propylene oxide, and the muscles were finally embedded in Epon. Thin ( 0 . 5 ~ sections
)
were stained with Richardson’s (1960) stain
and were photographed in bright field illumination on a Zeiss photomicroscope. Ultrathin
sections (0.06~)were stained with uranyl acetate and lead citrate and were subsequently
examined with a n Hitachi model HU-11-C
electron microscope.
Solutions used
The physiologic saline contained: 125 mM
NaCl; 2.7 mM KC1; 25 mM Tris-HC1, pH 7.5;
1.8 mM CaCl,; 0.5 mM MgCl,; 0.12 mM glucose.
MUSCLE-TENDON JUNCTION
295
to generate tension when provided with
MgATP (Fig. 3). Extraction also leaves functionally intact the mechanical junction between the contractile apparatus and the tendon, as is indicated by the ability of the tendon
to transmit tension from the extracted muscle
fiber to the force transducer. That this force is
generated totally by the muscle is shown by
the complete lack of response of the tendon to
ATP.
Electron microscopy of the extracted muscles
reveals that the plasma membrane (as well as
the membranous organelles of the sarcoplasm)
are removed by the detergent (Figs. 4, 5),
whereas the basal lamina retains its structure
and its spatial relationship to the cell periphery. The thin (actin) filaments end in a dense
layer which is composed of dense globules in
a less dense background (Figs. 4, 5). A slight
indication of a similar organization can also
Materials
be seen in unextracted fibers (Fig. 2). This
All chemicals were reagent quality. Di- dense layer is connected to the lamina densa
thiothreitol, phenylmethylsulfonyl fluoride, of the basal lamina by fine filaments which
PIPES, Tris, ATP, imidazole and Triton X-100 traverse the lamina lucida and the space forwere purchased from the Sigma Chemical merly occupied by the lipoprotein bilayer of the
plasma membrane. The filaments do not stain
Company.
well with uranyl acetate and lead citrate; nor
RESULTS
is this staining enhanced by using fixation proMurine extensor carpi radialis longus and tocols employing alcian blue, ruthenium red,
brevis muscles were chosen for this study be- cetylpyridinium chloride, or tannic acid. Meascause (a) they are fusiform muscles with dis- urements of the filament diameters are actinct myotendinous junctions and long ten- cordingly uncertain, but are estimated to fall
in the range of 2 to 7 nm. The filaments cross
dons; (b) they are small enough to permit
convenient fixation, embedment, and section- the lamina lucida and the extracted membrane
ing through the entire thickness of the mus- space without interruption. Their orientation
cles; and (c) they are large enough to produce is more or less perpendicular to the long axis
of the actin filaments, but deviations from the
measurable tension using techniques readily
available. A light micrograph of an extracted perpendicular are frequent.
In most cases, the dimensions of the lamina
muscle (Fig. 1)shows that the muscle fiber and
the connective tissue mutually interdigitate at lucida and lamina densa are not appreciably
the myotendinous junction. Electron micro- altered by detergent extraction in the presence
of EGTA. However, in some instances the lamgraphs (Fig. 2) of the myotendinous junction
ina lucida is greatly widened (Fig. 6), and in
of an unextracted (control) muscle show that
the junction is characterized by the subplas- these instances the filaments which connect
malemmal density into which thin (actin) fil- the lamina densa to the actin-filament-density
aments appear to insert, a continous plasma are greatly lengthened. In extreme cases, the
membrane, and a basal lamina composed of a muscle fiber separates completely from the
lamina densa, which remains tightly adherent
30-nm thick lamina densa and a 20-nm thick
lamina lucida. The lamina lucida appears to to the connective tissue matrix (Fig. 7). The
be traversed by fine filaments connecting the widening of the lamina lucia and the complete
plasma membrane to the lamina densa. Ex- separation of myofiber from lamina densa octernal to the lamina densa is a filamentous cur to a lesser extent in a modified extraction
solution, from which the EGTA is omitted.
matrix.
Extraction of the muscles with Triton X-100 However, if the “extraction solution” is releaves the contractile apparatus intact, as is placed by the “calcium extraction solution,’’
shown by the ability of the extracted muscles which contains 1.5 mM CaCl,, the myofibers
The Extraction solution contained: 1% v/v
Triton X-100;2 mM MgC12; 2 mM EGTA; 10
mM PIPES. NaOH, pH 6.0; 50 mM KC1; 1 mM
DTT; 1 mM PMSF.
The calcium extraction solution contained:
1% Triton X-100;2 mM MgC1,; 1.5 mM CaC1,;
10mM PIPES, pH 6.0; 50 mM KCl; 1 mM DTT;
1 mM PMSF.
The equilibration solution contained: 10mM
Imidazole-HC1, pH 7.0; 2 mM MgC1,; 50 mM
KC1; 1 mM DTT.
The contracting solution contained: 10 mM
Imidazole-HC1, pH 7.0; 2 mM MgC1,; 0.1 mM
CaC1,; 1.5 mM ATP; 50 mM KC1; 1 mM DTT.
Abbreviations used: DTT; Dithiothreitol;
PIPES; piperazine-N,N’-bis (2-ethanesulfonic
acid); EGTA; ethylene glycol-bis-(B-aminoethy1 ether) N,N‘-tetraacetic acid; PMSF; phenylmethylsulfonyl fluoride.
296
J.A. TROTTER, K. CORBETT, AND B.P. AVNER
297
MUSCLE-TENDON JUNCTION
do not separate from the lamina densa, nor
does the lamina lucida show any indication of
widening.
In regions other than the myotendinous
junction, the periphery of detergent-extracted
myofibers is characterized by a distinct space
separating myofibrils from basal lamina (Fig.
8). The width of this space is quite variable
(up to several microns) and contains a variable
amount of electron-dense material in the form
of globules (-- 20 nm in diameter), flocculent
material, and filaments (6-13 nm). Similar regions of unextracted fibers show that this space
is occupied by mitochondria and other membranous structures, and by ribosomes in a
dense, vaguely filamentous, matrix (Fig. 9).
The dense material juxtaposed to the plasmalemma in the unextracted cell (Fig. 9)has
apparent connections to the basal lamina in
the extracted cells (Fig. 8). The dense material
is sometimes seen to form transverse plates in
register with the Z lines of the most peripheral
myofibrils (not illustrated).
DISCUSSION
The results presented here establish that
exposure of skeletal muscle to Triton X-100for
48 hr results in the complete elimination of
morphologically identifiable membranes, both
plasma membrane and internal membranes.
Since no chemical studies have been performed, it is impossible to specify the extent
to which various membrane components have
been extracted. It seems prudent, therefore, to
consider the effect of Triton X-100 on muscle
to be the destruction of the integrity of membranes, rather than the complete extraction of
membrane components.
Indeed, the ability of extracted muscles to
transmit force to their tendons argues forcefully that a t least one component of the plasmalemma is not extracted by Triton X-100:
Fig. 1. Light micrograph of the muscle-tendon junction
of a fiber (F) of the extensor carpi radialis longus muscle.
T, tendon; N, nucleus of fibrocyte. A region comparable but
not identical to that enclosed in the dotted square is shown
in Figure 2. x 1,600.
Fig. 2. Electron micrograph of a portion of the
muscle-tendon junction region of an unextracted (control)
muscle. The lamina lucida (L) is populated by numerous
fine filaments, many of which appear to connect the plasma
membrane (open arrowhead) with the lamina densa (D). The
cytoplasmic surface of the plasma membrane is characterized by a dense layer (with faintly visible globules) (arrowhead) into which the thin filaments appear to insert. x
97,000.
namely, that element (or elements) which mechanically couples the contractile proteins to
the extracellular tensile structures. The fact
that Triton X-100is known to be very effective
as a solvent of membrane lipids, and very ineffective as a disrupter of protein-protein interactions (Helenius and Simons, 1975),
strongly suggests that proteins which span the
hydrophobic domain of the muscle membrane
are the elements which form this mechanical
link. The number of putative polypeptides
forming this linking structure is not determinable from these studies. There may be a
single transmembrane polypeptide, analogous
to Band 3 in the erythrocyte membrane (Lux,
1979),which binds to contractile proteins on
the cytoplasmic side of the membrane and to
extracellular macromolecules on the other. Or
there may be several polypeptides which interact within the plane of the membrane. Further studies will be required to explore these
possibilities.
The ultrastructural analysis of detergentextracted muscle-tendon junctions tends to
corroborate this view. Although a morphol-
2 MIN
T
t t
3
A G
Fig. 3. Force generated by detergent-extracted muscles
plotted versus time. In the upper tracing (M) the sutures
were both tied to muscle; therefore, this graph displays the
force generated by the myofibrils. In the middle tracing
(MT), one suture was tied to the tendon; this graph thus
displays the force transmitted from myofibrils to tendon. In
the bottom tracing (T),both sutures were tied to tendon. At
point “ A , ATP was added; a t point “ G , glutaraldehyde was
added. The drop in tension upon addition of glutaraldehyde
is inconsistent from experiment to experiment and therefore
cannot be interpreted.
298
J.A. TROTTER, K. CORBETT, AND B.P. AVNER
Figs. 4 and 5. Electron micrographs of detergent-extracted muscle-tendon junctions. The myofilaments of the
muscle fibers (F) are well preserved, a s is the basal lamina.
No cell membrane is seen. The lamina ludica (L) is crossed
by many fine filaments that run between the peripheral
density of the sarcoplasm and the lamina densa (D). Fine
arrows indicate regions in which the fine filaments are especially well seen; thick arrows indicate regions in which
thicker filaments are observed. The sarcoplasm has a globular appearance, especially in the peripheral regions. x
97,000.
299
MUSCLE-TENDON JUNCTION
Fig. 6. In the presence of EGTA, the lamina densa of
some detergent-extracted muscle-tendon junctions is partially separated from the sarcoplasm by a greatly widened
lamina lucida (L). Fine filaments are seen crossing the lam-
Fig. 7. This muscletendon junction, extracted in Triton
X-100 in the presence of EGTA, has separated almost completely. The lamina densa (D) has remained adherent to the
connective tissue, in which collagen (C) fibrils are seen. The
:-"i . , ~ , i ~av nnn
rln-mn
nlnh..la,
-ot..m
n C thn t n r m k - 1
rnrnnn1o.m
;o
.smll
300
J.A. TROTTER, K. CORBETT, AND B.P. AVNER
Fig. 8. A nonterminal portion of n detergent-extracted
muscle fiber shows a gap between the myofibrils (F) and the
lamina densa (D). This gap is occupied by varying amounts
of sarcoplasmic material composed of granular and filamentaus components. x 40.000.
Fig. 9. A nonterminal region of an unextracted muscle
shows that the space between the myofibrils and the plasma
membrane is occupied by membranes, mitochondria (M),
and ribosomes. X 40,000.
MUSCLE-TENDON JUNCTION
ogically recognizable plasmalemma is missing,
structural continuity between intracytoplasmic electron-dense material and the lamina densa is maintained by a population of fine
filaments. Filaments which extend between
the lamina densa and the plasmalemma at the
myotendinous junction have been previously
described (Hanak and Bock, 1971; Korneliussen, 1973; Ajiri, 1978). However, the present
work extends previous observations in two significant ways: (1)It demonstrates that the filaments of the lamina lucida are linked to cytoplasmic structures, either by directly passing
through the plasma membrane or by being
directly connected with transmembrane elements; and (2) it suggests that these filaments
actually carry tension, since both they and the
ability to transmit tension to tendons are preserved by Triton X-100 extraction of the muscles.
The smallest filaments observed in the extracted lamina lucida are about 2 nm, and the
largest are about 7 nm. There may be several
populations of filaments present in these regions; or, alternatively, the filaments may
have a tendency to aggregate, either naturally
or as a result of experimental intervention.
The filaments may also possess, or they may
be part of a filament complex that possesses,
considerable extensibility after a lengthy (48
hr) exposure to EGTA. Stretched and broken
filaments are observed when the lamina lucida
is moderately widened (up to 60 nm). That is,
a filament length in excess of 60 nm has not
been observed. When the lamina lucida exceeds 60 nm, as in the case of complete muscle-tendon separation, the filaments are absent, presumably because they have broken.
Inclusion of CaC12 during extraction of the
muscles prevents this separation of basal lamina from sarcoplasm. Although it is impossible
to identify the sites which are stabilized by
Ca2+,it is reasonable to think that they are
normally external to the outer leaflet of the
plasma membrane, since this locale is exposed
to the extracellular concentration of Ca2+(1.5
mM). It is probable, in any case, that a Ca2+sensitive site is involved in the linking of the
lamina densa to myofilaments, but not in the
junction between lamina densa and extracellular matrix. The Ca2+-sensitivesite might be
within the lamina densa. This notion is
strengthened by our recent observation that
in cultured myotubes the linkages between
myofibrils and the substratum are insensitive
to the concentration of Ca2+(unpublished observations).
301
“Microfibrils”(Hanack and Bock, 1971; Ajiri
et al., 19781,“thread-like or spine-like profiles”
(Korneliussen, 1973), and “filamentous structures” (Nakao, 1976) in the lamina lucida of
the myotendinous junction; and, “pillars or
partitions” or “strands” have been described
in the corresponding layers of desmosomes
(Kelly, 1966). The character and function of
these structures has remained speculative,
however. The present study, by showing a correlation between the presence of the fine filaments and of a functionally intact myotendinous junction, strengthens the idea that they
serve as mechanical linkers in the
muscle-tendon junction.
About the physical and chemical character
of the filaments, we have little information. It
is worth noting that the protein microfibrils
associated with elastic filaments are approximately 8 nm in diameter, and may be composed
of finer filaments, approximately 1.5 nm (Gotte
and Serafini-Fracassini, 1963) to 2.5 nm (Serafini-Fracassini, 1978). The triple helix of collagen also has a diameter of approximately 1.4
nm and associates to form fibrils of 8 nm and
greater (Eyre, 1979), which are probably composed of multiple repeats of 8-nm units (Parry
and Craig, 1979). Since the lamina densa of
basal laminae is composed in large part of type
IV collagen (Kefalides, 1980) and is morphologically composed of randomly arranged fibrils
in a granular matrix, the possibility exists that
the connecting fibrils described herein are in
part composed of basal lamina collagen. Carrying this speculation further, one could suggest that the transmembrane components
which are bound intracellularly to actin (either
directly or indirectly) are bound extracellularly to collagen microfibrils in the lamina lucida. These microfibrils might then coalesce to
form fibrils that are in turn cross-linked to the
collagen fibrils of the lamina densa. The crosslinked random filament structure of the lamina densa would be expected to provide the
system with a certain amount of rubbery elasticity (Alexander, 1968). Again, we have no
evidence at this time that the filaments of the
lamina lucida are collagen or elastin. However,
the extensibility of the filaments in the lamina
lucida suggests that they might be an elastic
element in series with myofilaments and tendon, and thereby serve as a damper during
contraction. A similar suggestion has been advanced by Hanak and Bock (1971).
Concerning the transmembrane component
of the linking system of the myotendinous
junction, we also have little information.
302
J.A. TROTTER, K. CORBETT, AND B.P. AVNER
Freeze-fracture studies on microvilli have
shown that 8-nm filaments which bind to intracellular actin filaments penetrate the lipid
bilayer (McNutt, 1978).In desmosomesas well,
traversing filaments have been described
which seem to bind to 10-nm filaments intracellularly and to pierce or traverse the membrane (Hull and Staehelin, 1979; Kelly and
Kuda, 1980).Also, we have recently described
transmembrane linking elements which apparently bind the actin of macrophages to extracellular substrata (Trotter, 1981). The nature of these structures is uncertain; in
particular, the number of molecules involved
is unknown. The filaments may be homo- or
heteropolymers of polypeptides or other macromolecules. In the myotendinous junction as
well, there is no evidence that a monomolecular filament extends from actin filaments to
the lamina densa. Rather, it seems more likely
that at least three distinct proteins are involved in the transmission of force: (1)A protein that binds actin and transmits tension to
the “membrane”; vinculin (Block and Geiger,
1980) is a possible candidate for this protein.
(2) A protein that spans the hydrophobic portion of the membrane. (3) A protein that transmits tension from the “membrane” to the lamina densa. At the moment, in terms of
chemistry, we are unable to do more than suggest possible candidates for these proteins.
Nonetheless, the fact that we can morphologically identify the tension-transmitting elements that are of physiological significance
encourages the belief that a molecular descrption of this region is possible.
ACKNOWLEDMENTS
The authors express appreciation to Ms. Suzanne Newel1 for her help in preparation of the
manuscript, and to Ms. Judi DeLongo for her
help in preparing thin and ultrathin sections.
This work was supported by a grant from the
Muscular Dystrophy Association.
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